This invention concerns screening systems and detection apparatus and methods for the inspection and analysis of multiple sample assays.
The invention concerns methods and apparatus for determining assay activity which results in the emission of photons and is of particular application to scintillation proximity assays, phosphorescence caused by the release of beta particles, liquid scintillation assays and luminescence assays (including chemi-luminescence, bio-luminescence and fluorescence), as well as cell-based assays, involving correlated bursts of photons. The invention is of particular application to assays in which the photon emission is relatively weak and in which discolouration or cloudiness in the liquid phase of the assay can inhibit the escape of photon emission to a detector. This interference with light transmission and/or reduction in efficiency in assay analysis is referred to as quench.
The invention is of particular application to the screening of multiple samples in pharmaceutical research and development using binding assay techniques and particularly scintillation proximity assays (SPA) using labelled beads or liquid scintillation counting using scintillation cocktails.
This invention may also be applied to correlated bursts of photons produced in luminescent assays, including cell based assays using luminescent labels indicators or substrates. These include enzyme linked assays, binding assay techniques and cell signalling assays (e.g. involving ion uptake and transport).
Historically photon detection has been by way of a photomultiplier tube (PMT). Such devices have a very fast rise time and high resolution in the time axis and can respond to photon emission durations of a few nanoseconds. Consequently if such emissions are occurring at intervals of a few microseconds, an electrical signal can be produced which is proportional to the light incident on the photocathode and a count rate (ie number of emissions per unit time) for a particular sample can be determined by counting the pulses from the PMT over a given window of time. A PMT based counter known as the TopCount (Registered Trade Mark) is produced by the Packard Instrument Company of USA. Techniques associated with the use of such counters for quench correction are described in a paper entitled Quench and Quench correction in Top Count topics published by the Packard Instrument Company in July 1993. Quench correction as applied to scintillation proximity assays is dealt with in a later paper also published by Packard Instrument Company under the heading Top Count Topics of February 1994. The use of a liquid scintillation counter in the presence of quench is also described in U.S. Pat. No. 4,633,088.
Since there is no X Y information attributable to the output signals from a photomultiplier tube (PMT), such a device can only be used to view one sample at a time. Where a large number of samples have to be inspected, either a very long period of time must be allowed so as to enable the PMT to be positioned successively over each of the samples for a sufficiently long period of time to allow a scintillation count to be determined for each sample, or in the other extreme a correspondingly large number of separate photomultiplier tubes must be mounted one above each of the samples so that all of the samples can be inspected simultaneously. In practice a compromise has normally been arrived at, and where the number of samples is of the order of 100 arranged in rows and columns (for example 8 rows and 12 columns), a row of eight PMT""s has been positioned above the array of samples so that the eight columns can be inspected simultaneously, and the row of PMT""s has been indexed row by row along the twelve columns. If each investigation is of one minute duration, the total assay inspection time will be something of the order of 12 minutes. In the example quoted, the total time if only one PMT were involved would be 96 minutes.
The 8xc3x9712 array of 96 samples is typical of so-called well plates but recently there has been a trend to larger well plates containing some hundreds of sample wells per plate and there is a desire to introduce even larger well plates having 1,000 or more wells per plate. Multiple sample assays using xe2x80x9cink dotxe2x80x9d techniques can have as many as 10,000 xe2x80x9cdotsxe2x80x9d per assay. The cost and complexity of using the correspondingly large number of PMT""s if assay times are to be kept low makes the manufacture of such an instrument unattractive and it is an object of the present invention to provide an alternative inspection system which is more sympathetic to large numbers of samples and which will allow overall assay inspection periods to be kept relatively short.
This last feature is of considerable importance where the chemistry of the assay is not sufficiently stable to allow a considerable period of time to elapse between the start and finish of an assay investigation.
Heavy demands are now being made on screening systems by developments in combinatorial chemistry and genomics. In these and other applications the phenomenon of quench can introduce errors. Quench occurs in liquid scintillation counting where specific components in the sample can interfere with the production and/or transmission of light. A reduced scintillation count results and unless steps are taken to correct for quench, it is impossible to say whether a low level of activity from a particular sample is due to low inherent photon emission (due for example to a lack of binding in the assay) or is due to the opacity of the liquid phase of the assay which inhibits the release of photons from what is otherwise a relatively active sample.
There are two recognised forms of quench.
Chemical quench occurs when the unwanted compound interferes with the scintillation or luminescence process causing non-radiative dissipation of energy. This reduces the apparent energy of the decay event and the number of photons produced.
Colour quench is an optical phenomenon whereby photons produced by the scintillation or luminescence process are absorbed by the unwanted material before reaching the detector.
This invention seeks to provide a detection system and method of inspection for use with microplates containing hundreds or thousands of individual reaction wells or multiple site assays containing many hundreds or thousands of reaction sites, and seeks to provide an apparatus and method which will speed up the inspection and analysis of such assays relative to the time which would be required using conventional techniques.
The invention also seeks to provide an improved detection system and method by which the apparent reduction in activity from any one site or well caused by discolouration or greying of the sample (colour quench); or by interference with the photon emitting properties of the assay by the sample ingredients or by-products (chemical quench); or both, can be corrected.
In particular, it is an object of the invention to provide a detection and counting system which can be adapted to accommodate variation due to quench, so that the numerical count obtained is substantially independent of quench.
According to one aspect of the present invention a detection system embodying the invention comprises:
1. means for supporting a multiple sample assay in an inspection station,
2. plural addressable photosensitive detector elements in an array which is positioned relative to the inspection station so that light emitted from a sample impinges on a unique group of photosensitive elements, to produce a change in the electrical characteristics thereof, so that a change which is attributable to the group is associated with that sample, which change is permanent at least until the detector elements have been addressed and any change in their characteristics has been read out as an item of information,
3. means for addressing the groups of elements at regularly occurring intervals of time to generate the said items of information,
4. means for attributing a numerical value to the individual items of information,
5. means for storing the numerical values in such a manner that all the values which relate to any one of the samples are linked,
6. means for storing a sample identifier for each set of linked values,
7. means for reading out the linked values relating to a sample to permit computational analysis thereof,
8. means for computing the arithmetic mean of the linked numerical values read out, and
9. means for supplying as output information the numerical value of the computed arithmetic mean of the linked values, together with the sample identifier therefor.
Where the light emitted per sample is very low, an image intensifier is preferably provided between the samples and the photosensitive detector elements. Typically an image intensifier CCD camera is employed. Alternatively a cooled CCD camera may be used operating at the higher frame rates now achievable with these devices, typically in the range 0.1-10 frames per second.
According to another aspect of the invention a method of analysing a multiple sample assay comprises:
1. locating the multiple sample assay in an inspection station,
2. imaging light emitted from the samples onto a plural array of addressable photosensitive detector elements so that light emitted from a sample impinges on a unique group of the photosensitive elements to produce a change in the electrical characteristics thereof so that a change attributable to the group is associated with that sample, which change is permanent at least until the elements have been addressed and any change in their characteristics has been read out as an item of information,
3. addressing the elements at regularly occurring intervals of time to generate the said items of information,
4. converting each item of information into a numerical value,
5. storing the numerical values in such a manner that all the values which relate to any one of the samples are linked.
6. generating and storing a sample identifier for each set of linked values,
7. reading out the linked values relating to a sample and computing the arithmetic mean of the linked numerical values, and
8. supplying as output information the numerical value of the computed arithmetic mean of the linked values, together with the sample identifier therefor.
Where the light emitted per sample is very low the method may include the step of image intensifying the light between the sample and the photosensitive detector elements, or a cooled CCD may be used.
The method may include the step of filtering the light between the samples and the detector element array.
The improved system and method is of particular use in assays in which the light emitting entity has a well defined probability of emitting a given number of photons per unit period of time, and in which there are many occasions when the entity emits two or more detected photons per disintegration. Examples of such assays in which the light emitting components of each sample have a well defined probability for emitting a given number of photons per initiating event are radio isotope labelled assays using for example Tritium or Carbon 14. Here there are equivalent well defined probabilities P0, P1, P2, P3 etc, of detecting 0, 1, 2, 3 . . . photoelectrons per radioisotope disintegration. The actual probabilities of course depend on the particular isotope, SPA bead (in the case of scintillation proximity assays); the scintillant; the optics; and the characteristics of the camera. Equivalent probabilities might be defined for the correlated emission of photons by a cell, proceeding via processes such as cell signalling, generation of ion fluxes, or enzyme activity, which are linked to luminescent signals.
Preferably at each addressing step each photosensitive detector element is reset to an initial state ready to begin receiving light again.
Preferably in such an arrangement the detector elements and operating conditions are selected so that the changes in electrical characteristics are linearly proportional or nearly so to the quantity of light incident thereon.
Preferably, before the system is used, a calibration step is performed in which samples of known activity are presented to the array of photosensitive elements, using an appropriate addressing time interval, so that output information from each sample-detector pair is available.
By using different samples of known concentrations of calibrated light emitting material such as Microspheres as produced by Arnersham International plc to vary the activity level in different samples, and performing a series of tests on these different known activities, so the observed numerical output information from the groups of detector elements in response to the different known activities can be logged against the known activity in a first calibration memory, to provide a look-up table for converting future numerical output information to sample activity.
Preferably each group of detector elements is subjected to a large number of exposures so as to obtain a large number of different outputs therefrom in each of the aforementioned tests before the mean numerical value attributable to any particular sample/detector group combination is computed.
Calibration may be speeded up by computing the mean value after each exposure and comparing each computed mean with the accumulating mean value (computed from all previous output information for the test from that detector group), and stopping that sequence of exposures and computations when the comparison indicates a desired level of similarity between the two computed mean values.
The process may be stopped for each detector group individually or may be continued until all the computed mean value comparisons for all the detector group has achieved the similarity criterion.
Any difference between the final accumulation mean value for each detector group and a theoretical expected mean value for the test may for example be stored as detector group response variations in a supplementary calibration memory.
In the preferred system, albeit requiring a large number of individually addressable photosensitive detectors in the detector array, each group contains a relatively large number of photosensitive detector elements so that light from the associated sample will now fall on some of this large number of adjacent photosensitive detector elements in the group.
A preferred photo detector element array is an image intensified integrating CCD. Such devices can be obtained with an array having a large number of individually addressable detector elements arranged in rows and columns.
For example using a 385xc3x97288 chip, when part of an 864 well plate is viewed, each of the wells can be typically associated with a respective 20xc3x9720 group of pixels on the camera chip.
Devices are available having nominally 3000 rows and nominally 2000 columns of photosensitive elements, ie a nominal total of 6,000,000 elements and such a device can permit up to 10,000 appropriately arranged samples to be imaged each sample having available to it a group of 25xc3x9725 pixels.
Thus in such a device, light emanating from a sample will result in some of the pixels in its associated 25xc3x9725 group of pixels changing state, and generating an appropriate output signal at the end of the frame interval as the camera chip is read out. The change of state of these pixels is caused by the detection of a single photon, and the flux of photons on the array can be sufficiently low for it to be very unlikely that these pixels will receive more than one photon during a single frame, even though a 25xc3x9725 group of pixels may receive many photons during a frame. Thus, the more light (i.e. the larger the number of emitted photons) that has emanated from a sample well the greater is likely to be the number of individual pixels in the corresponding 25xc3x9725 array which will have registered incident light, and therefore the greater the number of xe2x80x9cchange of statexe2x80x9d transitions which will be counted at the end of that frame interval for that sample to be linked with the sample identifier.
By way of example the input area of a 40 mm camera is typically 32xc3x9724 mm2, but in accordance with another feature of the invention this can be increased using optical devices based on lenses or fibre optic tapers or both, so as to receive light from a sample plate occupying a larger area.
A filter may be incorporated between the samples and the image intensifier to remove unwanted wavelengths.
The separate pixels in each 25xc3x9725 group can be individually addressed, and since the likelihood of light from any one sample impinging on the same pixel in the relevant group during a typical frame period of 10 milliseconds is very low, the individual detector elements of a CCD camera chip can be considered to be substantially bistable devices which are reset at the end of each frame period, and whose state at the end of each exposure (frame) is either low or high depending on whether or not light has impinged thereon. If low, light energy has been incident on the pixel and when it is scanned during read-out, an output information signal indicating this is produced as each such pixel is reset. Using an integrating CCD camera chip having a defined frame period (ie interval between read-outs) each group of pixels will acquire light from its related sample during each frame period and if it is assumed that r photoelectrons are detected per frame by the group (where r is greater than or equal to zero) then at the end of each frame the frame store associated with the chip is provided with another value of r for that group. Since each group corresponds to one of the samples in the array under analysis, a sample identifier can be associated with each of the string of values of r from each group, which is available to be linked with the mean value computation for the string of values r.
Hence, the number of photons emitted from a sample region per frame is measured for each of a succession of (for example 5000) frames. This enables a frequency distribution of detected photons to be calculated. It will be appreciated that, in this context, the term xe2x80x9cfrequencyxe2x80x9d refers to the number of photons emitted per unit time, not the intrinsic frequency of each photon.
Preferably a CCD camera chip is selected which employs charge transfer thereby speeding up the read-out of the array. In such devices a second xe2x80x9chiddenxe2x80x9d array of elements is provided in addition to the primary array, and the charge condition in each row of the first array is transferable in parallel into a corresponding row in the second array on receipt of a reset signal at the end of each frame interval, the reset signal serves to restore the charge in each of the detector elements of the primary array and this allows the whole of the following frame interval for reading out the charge pattern of the secondary array.
The first calibration step described above, allows a look-up table to be created of sample activity against mean numerical value per sample-group pair. This calibration step is necessary before a CCD camera can be used on unknown assay samples.
Second and third calibrations are also necessary if quench can affect future (unknown) assay samples to be imaged and analysed.
The first of these additional calibrations allows activity level measurement to be corrected for colour quench. The look-up table needed is compiled as follows:
Using an imaging device as aforesaid, a large number of exposures (frames) are performed using samples of constant (known) activity and having different (known) dye concentrations, and from each set of results the mean number of detected photons per frame from a sample (M1) and the variance (V1) about the mean are computed and a first Index (I1) is computed using the formula (V1xe2x88x92M1)/M1. The computed value of I1 for each different dye concentration allows the look-up table to be compiled, for colour quench correction.
The mean and variance and formula for computing the index may be adjusted to compensate for random background photons, as occur with phosphorescence from the sample plate background and with the intrinsic autofluorescence found in cell-based assays.
The other additional calibration allows activity level measurement to be corrected for chemical quench, and another look-up table is created in the same way. Thus samples of constant (known) activity having different (but known) concentrations of a chemical quenching agent such as nitro-methane are imaged over many frames, and the mean (M2) and variance (V2) of the results are computed to provide a second index I2 for different nitro-methane concentrations, to generate a chemical quench correction look-up table, using the formula I2=(V2xe2x88x92M2)/M2.
The xe2x80x9cColour quench correctionxe2x80x9d look-up table constitutes the second calibration, and the xe2x80x9cChemical quench correctionxe2x80x9d look-up table constitutes the third calibration referred to above.
Disregarding quench (and other factors which can affect the number of photons emitted in response eg to radio isotope disintegration), to a first approximation the change in charge in any photosensitive elements in a CCD camera array and hence the number of detected photons will be a measure of the radioisotope or luminescent activity in the sample. Where this measured activity is affected by the underlying process being determined in the sample (eg binding) then this activity will also therefore correspond to the degree of binding which is occurring in the sample which is imaged thereon. In general relatively small amounts of light will be emitted from a sample in response to each radioisotope disintegration and these will on average occur at relatively well spaced intervals, although lasting individually for very short periods of time.
The photon emission from a single Tritium disintegration per sample per CCD exposure period typically will result in only a handful of detected photoelectrons (typically in the range 1-10). The number can be increased using an image intensifier before being supplied to a CCD chip.
Using a cooled CCD, instead of an image intensified CCD camera, only the handful of photoelectrons would be available to the CCD chip, but by operating such a cooled CCD camera in the range 0.1-10 frames per second, there may be sufficient information available to allow the present invention to be performed, as the output signals from the CCD.
Where higher photon producing assays are used the intensifier may be gated, so as to reduce the number of photoelectrons reaching the CCD chip or image intensifier CCD camera to prevent saturation or damage.
For each gate setting the camera or intensifier-camera combination can be calibrated by using samples of known activity.